Although previous studies from this group have indicated the absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for virus replication, this enzymatic function has received little attention as an antiviral target. However, the finding that HIV-1 RNase H and integrase (IN) are derived from the same superfamily of nucleotidyl transferases has allowed the implementation of screening strategies based on IN inhibitors previously identified at the NCI. Several natural products have initially been identified that act on both RNase H and IN, while a limited number show selectivity for the individual proteins. Subsequently, two fluorescence-based assays have been implemented in collaboration with the NCI Molecular Targets Development Program for high throughput screening of several NCI libraries (n = 250,000) for inhibitors of HIV-1 RNase H function (Parniak et al., 2003, Chan et al., 2004). Several classes of inhibitors, active at sub-micromolar concentrations, have been identified with enhanced specificity for HIV-1 RNase H. Presently, the binding site for inhibitors on the HIV-1 E.coli and human RNases H is being determined by both X-ray crystallography and high resolution mass spectrometry. A patent on a selected group of compounds with antiviral activity has also been submitted.